Most eukaryotes harbor a high proportion of transposable elements (TEs) in their genomes. Heterochromatin, a condensed chromatin state found at domains enriched for TEs and other repetitious elements, is important for silencing TEs and maintaining the integrity of the genome. Significant portions of eukaryotic genomes, including the Y chromosome, are heterochromatic, made up largely of repetitive sequences and possessing a distinctive chromatin structure associated with gene silencing. Heterochromatic regions have a high repeat content and are characterized by specific histone modifications, but the primary sequence elements that define specific chromosomal domains as preferred sites of heterochromatin assembly are not well understood. In addition, recent work has shown that the composition and organization of Drosophila heterochromatin is spatially heterogeneous and dynamic, and a variety of cellular pathways and molecular components create genetically inert heterochromatin, but only a subset of these components has been characterized. Our proposal aims to dissect the cis- and trans-acting cellular mechanisms involved in heterochromatin formation, by studying the genome-wide establishment of heterochromatin during early development in different wildtype and transgenic Drosophila strains and species. Recent studies suggest that small RNAs ?? possibly derived from transposable elements (TEs) ?? or specialized DNA-binding zinc finger proteins contribute to heterochromatin targeting. Using a combination of comparative sequence analysis, gene expression studies, small RNA profiling and ChIP-seq experiments across development to map histone modifications associated with heterochromatin and genome interaction maps, we will characterize the spatiotemporal heterogeneity of genome-wide heterochromatin establishment across development in Drosophila melanogaster and D. miranda, and catalog the establishment and maturation of inert chromatin in 3D during early development. This will reveal which sequences serve as nucleation sites for inactive chromatin, and how silencing chromatin spreads across the genome. The initial establishment of heterochromatin is driven by RNA and protein components that are maternally deposited into the fertilized egg. We will utilize the wealth of D. melanogaster resources to study heterochromatin formation in early embryos by depleting maternally deposited candidate genes involved in establishing heterochromatin in the developing embryo. Integrating our results across aims will provide a full picture of how heterochromatin is established in the early embryo, whether the initial establishment of closed chromatin proceeds in a sequential manner, and how it spreads across the repetitive regions of the genome. We will dissect the heterogeneous and dynamic composition of Drosophila heterochromatin across development and in embryos where components of the heterochromatin pathway are depleted using transgenic approaches. This will provide a full picture of the various molecular pathways and different molecular components involved in creating genetically inert heterochromatin.